CN114176008A - Tissue culture breeding method for notopterygium incisum seeds - Google Patents
Tissue culture breeding method for notopterygium incisum seeds Download PDFInfo
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Abstract
The invention discloses a culture medium for tissue culture and breeding of notopterygium incisum seeds and a tissue culture and breeding method of the notopterygium incisum seeds. Experiments prove that the survival rate of the tissue culture seedlings obtained by the tissue culture breeding method of notopterygium incisum seeds after transplantation reaches over 90 percent, lays a solid foundation for the large-scale propagation of notopterygium incisum, and has popularization and application values.
Description
Technical Field
The invention relates to the field of plant tissue culture, in particular to a tissue culture breeding method for notopterygium incisum seeds.
Background
Notopterygium (Notopterygium inccisum Ting ex H.T.Chang) is a perennial herb of Notopterygium (Notopterygium) of the family Umbelliferae, which is unique in China. The medicinal part of notopterygium root recorded in pharmacopoeia is dry rhizome and root, the main chemical components comprise compounds such as notopterygium alcohol, isoimperatorin and the like, and the pharmaceutical composition has the effects of relieving exterior syndrome, dispelling cold, dispelling wind, removing dampness and relieving pain, and is used for treating wind-cold type common cold, headache and neck weakness, rheumatic arthralgia and shoulder and back ache. The notopterygium root is a prescription medicinal material of a plurality of famous patent medicines, wherein the classic medicine for dispelling wind cold is a Jiuwei notopterygium root pill, and the classic medicine for dispelling wind and removing dampness is a notopterygium root dampness-eliminating decoction. Due to the good medicinal effect of notopterygium root, notopterygium root is popular with people and has good market value.
Notopterygium incisum grows in high-altitude areas, the production area is cold and humid, the ecological environment is severe, the Notopterygium incisum grows slowly, the seed development is not complete enough, and the Notopterygium incisum has a very long dormancy stage. Fangziseng et al performed multiple field investigations on wild Notopterygium incisum in Gansu, and found that the natural seed reproduction rate under the natural conditions of wild Notopterygium incisum in Gansu is only 0.52%. Meanwhile, researches find that notopterygium incisum seeds have morphological physiological dormancy, the notopterygium incisum seeds can germinate in two years under natural conditions, and the viability of the seeds is reduced along with the time extension, so that notopterygium incisum is difficult to propagate in the natural environment. Notopterygium incisum can also be propagated by rhizomes, but a large amount of medicinal materials are consumed, the propagation coefficient is low, and the price is high.
With the development of plant tissue culture technology, a great deal of propagation of more and more plants in a short time is realized under the support of the tissue culture technology. Patent CN108719072A discloses a tissue culture and rapid propagation technology of notopterygium incisum seeds, which solves the problems of long culture period and low propagation coefficient of notopterygium incisum. However, plant seeds are cultivated by using a tissue culture medium, the survival rate after transplanting is low due to the problems of root induction rate, seedling rate, growth vigor of a tissue culture stage and the like, and finally, a large amount of plants cannot be expanded.
Disclosure of Invention
In order to solve the problems, the invention provides a culture medium for tissue culture and breeding of notopterygium incisum seeds, which comprises a germination culture medium, a cluster seedling induction culture medium and a root system induction culture medium;
the germination culture medium is a culture medium which takes an MS culture medium as a basic culture medium, is added with a plant growth regulator with the final concentration of 0.1-4.2 mg/L, agar with the final concentration of 5-10 g/L and cane sugar with the final concentration of 25-35 g/L, and has the pH value of 5.5-6.0;
the cluster seedling induction culture medium is a culture medium which takes 1/2MS as a basic culture medium, is added with a plant growth regulator with the final concentration of 1-4.5 mg/L, agar with the final concentration of 5-10 g/L and sucrose with the final concentration of 25-35 g/L, and has the pH value of 5.5-6.0;
the root system induction culture medium is a culture medium which takes 1/2MS culture medium as a basic culture medium, is added with plant growth regulator with final concentration of 0.4-1 mg/L, agar with final concentration of 5-10 g/L and sucrose with final concentration of 15-25 g/L, and has pH value of 5.5-6.0.
Further, the plant growth regulator is any one or more of 6-benzylpurine (6-BA), alpha-naphthylacetic acid (NAA), indolebutyric acid (IBA), gibberellin (GA3) and 2, 4-Epibrassinolide (EBR).
Furthermore, the germination culture medium is a culture medium which takes an MS culture medium as a basic culture medium, is added with 6-benzylpurine (6-BA) with the final concentration of 1.0mg/L, alpha-Naphthalene Acetic Acid (NAA) with the final concentration of 2.0mg/L, agar with the final concentration of 8g/L and sucrose with the final concentration of 30g/L and has the pH value of 5.8;
the clustered seedling induction culture medium is a culture medium with a pH value of 5.8, wherein 1/2MS culture medium is used as a basic culture medium, and 6-benzylpurine (6-BA) with a final concentration of 0.3-1 mg/L, indolebutyric acid (IBA) with a final concentration of 0.6mg/L, gibberellin (GA3) with a final concentration of 0.2mg/L, agar with a final concentration of 8g/L and sucrose with a final concentration of 30g/L are added;
the root system induction culture medium is a culture medium which takes 1/2MS culture medium as a basic culture medium, is added with indolebutyric acid (IBA) with the final concentration of 0.6mg/L, agar with the final concentration of 8g/L and cane sugar with the final concentration of 20g/L, and has the pH value of 5.5-6.0.
Further, the germination culture medium is a culture medium which takes an MS culture medium as a basic culture medium, is added with 2, 4-Epibrassinolide (EBR) with the final concentration of 0.1mg/L, agar with the final concentration of 8g/L and sucrose with the final concentration of 30g/L and has the pH value of 5.8;
the clump shoot induction culture medium is a culture medium which takes 1/2MS culture medium as a basic culture medium, is added with 6-benzylpurine (6-BA) with the final concentration of 1.5mg/L, alpha-naphthylacetic acid (NAA) with the final concentration of 2.0mg/L, indolebutyric acid (IBA) with the final concentration of 0.6mg/L, gibberellin (GA3) with the final concentration of 0.2mg/L, agar with the final concentration of 8g/L and sucrose with the final concentration of 30g/L, and has the pH value of 5.8;
the root system induction culture medium is a culture medium which takes 1/2MS culture medium as a basic culture medium, is added with indolebutyric acid (IBA) with the final concentration of 0.6mg/L, alpha-naphthylacetic acid (NAA) with the final concentration of 0.2mg/L, agar with the final concentration of 8g/L and cane sugar with the final concentration of 20g/L and has the pH value of 5.5-6.0.
Further, the germination medium is a medium with the pH value of 5.8, which takes an MS medium as a basic medium and is added with 6-benzylpurine (6-BA) with the final concentration of 1.0mg/L, alpha-Naphthalene Acetic Acid (NAA) with the final concentration of 3.0mg/L, gibberellin (GA3) with the final concentration of 0.2mg/L, agar with the final concentration of 8g/L and sucrose with the final concentration of 30 g/L;
the clump shoot induction culture medium is a culture medium with 1/2MS as a basic culture medium, added with 6-benzyl purine (6-BA) with the final concentration of 1.5mg/L, indolebutyric acid (IBA) with the final concentration of 0.6mg/L, gibberellin (GA3) with the final concentration of 0.2mg/L, agar with the final concentration of 8g/L and cane sugar with the final concentration of 30g/L, and the pH value is 5.8;
the root system induction culture medium is a culture medium which takes 1/2MS culture medium as a basic culture medium, is added with indolebutyric acid (IBA) with the final concentration of 0.6mg/L, alpha-naphthylacetic acid (NAA) with the final concentration of 0.2mg/L, agar with the final concentration of 8g/L and cane sugar with the final concentration of 20g/L and has the pH value of 5.5-6.0.
Further, the germination culture medium is a culture medium which takes an MS culture medium as a basic culture medium, is added with 2, 4-Epibrassinolide (EBR) with the final concentration of 0.1mg/L, agar with the final concentration of 8g/L and sucrose with the final concentration of 30g/L and has the pH value of 5.8;
the clump shoot induction culture medium is a culture medium with 1/2MS as a basic culture medium, added with 6-benzyl purine (6-BA) with the final concentration of 1.5mg/L, indolebutyric acid (IBA) with the final concentration of 0.6mg/L, gibberellin (GA3) with the final concentration of 0.2mg/L, agar with the final concentration of 8g/L and cane sugar with the final concentration of 30g/L, and the pH value is 5.8;
the root system induction culture medium is a culture medium which takes 1/2MS culture medium as a basic culture medium, is added with indolebutyric acid (IBA) with the final concentration of 0.6mg/L, alpha-naphthylacetic acid (NAA) with the final concentration of 0.2mg/L, agar with the final concentration of 8g/L and cane sugar with the final concentration of 20g/L and has the pH value of 5.5-6.0.
The invention finally provides a tissue culture breeding method of notopterygium incisum seeds, which utilizes the culture medium for cultivation and comprises the following specific steps:
a. taking notopterygium incisum seeds, stacking in wet sand at 20-30 ℃ for 2-4 months, stacking in wet sand at 2-8 ℃ for 2-4 months, cleaning, germinating for 1 month, taking non-germinated plump seeds, stripping episperm, cleaning, and sterilizing to obtain sterile seeds
b. Taking sterile seeds, cutting out wounds, inoculating the sterile seeds into a germination culture medium, and culturing to obtain sterile seedlings;
c. taking the aseptic seedlings in the step b, cutting off cotyledons, and then transferring the aseptic seedlings into a clump seedling induction culture medium for culture to obtain clump seedlings;
d. dividing the cluster seedlings into single plants, inoculating the single plants into a root system induction culture medium, and culturing to obtain notopterygium root tissue culture seedlings.
Further, the cleaning and disinfection in the step a are carried out by soaking in soap water for 1min, washing with running water for 30min, finally soaking in 75% ethanol for 20s, cleaning with sterile water, soaking in 0.1% mercuric chloride for 9min, and cleaning with sterile water.
Further, the germination in the step a is carried out for 1 month under the conditions that the temperature is 15 ℃, the humidity is 75%, the light intensity is 2200Lux, and the light cycle is 12h (light)/12 h (dark).
Further, the stratification was for 3 months.
Further, the wound of step b is on the side of the seed edge.
Further, 2-4 leaves are reserved on the single plant in the step d.
Further, the culture conditions are as follows: the temperature is 23 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 12h (light)/12 h (dark).
The 1/2MS culture medium is a culture medium with half of macroelements in the MS culture medium formula and unchanged content of other components.
The MS culture medium is one of the most common culture media in plant tissues, and the formula of the MS culture medium comprises the following components:
compared with the prior art, the invention has the following remarkable effects:
1) the notopterygium incisum seeds which do not germinate after dormancy breaking are used, so that the utilization rate of the notopterygium incisum seeds is improved, and resource waste is avoided;
2) by adopting a method of treating seeds by mechanical damage, notopterygium incisum seeds start to germinate on day 4, so that the germination time of the seeds is shortened, and the germination rate of the seeds is improved;
3) the preferred culture medium for inducing the germination of the seeds improves the germination rate of the mechanically treated seeds to 70.48 percent;
4) the culture process of forming callus through dedifferentiation is not carried out, so that the occurrence of variation seedlings which are easy to generate in the growth process of the callus is avoided, and the genetic stability of the excellent characters of notopterygium root is ensured;
5) the preferable temperature and illumination can ensure the good growth of the notopterygium tissue culture seedlings, improve the photosynthesis of notopterygium leaves and avoid the phenomenon of leaf browning and death;
6) the cluster seedling induction culture medium can effectively induce and differentiate cluster seedlings, and the propagation coefficient of aseptic seedlings is 5.62 at most;
7) the rooting culture medium has a good induction effect on the rooting of the notopterygium root tissue culture seedlings, the root induction rate of the rooting culture medium reaches 17.39%, the root system is strong and divergent, the tissue culture seedlings which do not root are good in growth condition, and part of the tissue culture seedlings continue to differentiate into clumpy seedlings. In the culture process, overground parts of notopterygium roots grow well, the seedling rate is up to 95%, and after-period tests prove that the survival rate of the tissue culture seedlings obtained by the notopterygium root seed tissue culture breeding method after transplantation reaches 95.83%, so that a solid foundation is laid for the large-scale propagation of notopterygium roots.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
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FIG. 1 sterile Germination of Notopterygium incisum seeds (a. sterile Germination of seeds in case of side cut; b. sterile Germination of seeds in case of cross cut; c. sterile Germination of seeds in case of longitudinal cut)
FIG. 2 shows the growth and development stages of Notopterygium incisum tissue culture plantlets (a. initial growth conditions of cluster bud induction, b. growth conditions of cluster buds cultured in a culture medium for 30d, c. growth conditions of tissue culture plantlets in rooting culture for 30d, d. growth conditions of tissue culture plantlets in hardening-seedling transplantation for 30 d)
Detailed Description
The raw materials, reagents and equipment used in the embodiment of the invention are all known products; may be obtained by commercial purchase.
Example 1 sterile Germination and Rapid propagation of Notopterygium incisum seeds
Step 1, pretreatment of notopterygium incisum seeds: the notopterygium seeds are firstly layered with wet sand at 25 ℃ for 3 months, then layered with wet sand at 4 ℃ for 3 months, taken out, washed clean with distilled water, and placed in a culture dish paved with filter paper for germination experiments in a constant-temperature illumination incubator with the temperature of 15 ℃, the humidity of 75%, the illumination intensity of 2200Lux and the illumination period of 12h (light)/12 h (dark). After 1 month, the ungerminated seeds were collected, mixed well and placed on moist sterile filter paper, which was stored in a refrigerator at 4 ℃.
Step 2, selecting and treating notopterygium seed explants: selecting healthy seeds, removing the testa of the seeds, soaking for 1min with soap water, and washing for 30min under running water;
step 3, sterilizing notopterygium seeds: soaking and disinfecting the notopterygium incisum seeds treated in the step 2 with 75% of ethanol for 20s on a sterile operating platform, then cleaning with sterile water for 2 times, then soaking and disinfecting with 0.1% of mercuric chloride for 9min, and then cleaning with sterile water for 6 times to obtain sterile seeds;
step 4, aseptically germinating notopterygium seeds: placing the sterile seeds obtained in the step 3 on sterile filter paper, cutting a wound on one side edge by using a scalpel, and inoculating the damaged seeds into a culture medium, wherein the germination culture medium comprises: MS culture medium, 1.0 mg/L6-benzylpurine (6-BA), 2.0mg/L alpha-Naphthalene Acetic Acid (NAA), 8g/L agar and 30g/L sucrose; the culture conditions of the germination culture medium are that the pH is 5.8, the temperature is 23 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 12h (light)/12 h (dark);
step 5, cluster seedling induction of aseptic seedlings: and (3) cutting off cotyledons on two sides of the sterile seedling of the seed germinated in the step (4) in a sterile operating platform, and transferring the sterile seedling into a cluster seedling induction culture medium, wherein the cluster seedling induction culture medium comprises: 1/2MS culture medium, 0.3 mg/L6-benzylpurine (6-BA), 0.6mg/L indolebutyric acid (IBA), 0.2mg/L gibberellin (GA3), 8g/L agar, 30g/L sucrose; the culture conditions of the germination culture medium are that the pH is 5.8, the temperature is 23 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 12h (light)/12 h (dark);
step 6, rooting induction of the tissue culture seedlings: dividing the cluster seedlings induced by the aseptic seedlings in the step 5 into single plants (each plant keeps about 3 leaves) in an aseptic operation table, and inoculating the single plants into a root system induction culture medium, wherein the root system induction culture medium comprises: 1/2MS culture medium, 0.6mg/L indolebutyric acid (IBA), 8g/L agar, 20g/L sucrose; the culture conditions of the germination culture medium are that the pH is 5.8, the temperature is 23 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 12h (light)/12 h (dark);
example 2 sterile Germination and Rapid propagation of Notopterygium incisum seeds
Step 1, pretreatment of notopterygium incisum seeds: the notopterygium seeds are firstly layered with wet sand at 25 ℃ for 3 months, then layered with wet sand at 4 ℃ for 3 months, taken out, washed clean with distilled water, and placed in a culture dish paved with filter paper for germination experiments in a constant-temperature illumination incubator with the temperature of 15 ℃, the humidity of 75%, the illumination intensity of 2200Lux and the illumination period of 12h (light)/12 h (dark). After 1 month, the ungerminated seeds were collected, mixed well and placed on moist sterile filter paper, which was stored in a refrigerator at 4 ℃.
Step 2, selecting and treating notopterygium seed explants: selecting healthy seeds, removing the testa of the seeds, soaking for 1min with soap water, and washing for 30min under running water;
step 3, sterilizing notopterygium seeds: soaking and disinfecting the notopterygium incisum seeds treated in the step 2 with 75% of ethanol for 20s on a sterile operating platform, then cleaning with sterile water for 2 times, then soaking and disinfecting with 0.1% of mercuric chloride for 9min, and then cleaning with sterile water for 6 times to obtain sterile seeds;
step 4, aseptically germinating notopterygium seeds: placing the sterile seeds obtained in step 3 on sterile filter paper, cutting the seeds into two halves along the ventral suture of the seeds by using a scalpel, and inoculating the seeds into a culture medium, wherein the germination medium comprises: MS culture medium, 1.0 mg/L6-benzylpurine (6-BA), 2.0mg/L alpha-Naphthalene Acetic Acid (NAA), 8g/L agar and 30g/L sucrose; the culture conditions of the germination culture medium are that the pH is 5.8, the temperature is 23 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 12h (light)/12 h (dark);
step 5, cluster seedling induction of aseptic seedlings: and (3) cutting off cotyledons on two sides of the sterile seedling of the seed germinated in the step (4) in a sterile operating platform, and transferring the sterile seedling into a cluster seedling induction culture medium, wherein the cluster seedling induction culture medium comprises: 1/2MS culture medium, 0.5 mg/L6-benzylpurine (6-BA), 0.6mg/L indolebutyric acid (IBA), 0.2mg/L gibberellin (GA3), 8g/L agar, 30g/L sucrose; the culture conditions of the germination culture medium are that the pH is 5.8, the temperature is 23 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 12h (light)/12 h (dark);
step 6, rooting induction of the tissue culture seedlings: dividing the cluster seedlings induced by the aseptic seedlings in the step 5 into single plants (each plant keeps about 3 leaves) in an aseptic operation table, and inoculating the single plants into a root system induction culture medium, wherein the root system induction culture medium comprises: 1/2MS culture medium, 0.6mg/L indolebutyric acid (IBA), 8g/L agar, 20g/L sucrose; the culture conditions of the germination culture medium are that the pH is 5.8, the temperature is 23 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 12h (light)/12 h (dark).
Example 3 sterile germination and Rapid propagation of Notopterygium incisum seeds
Step 1, pretreatment of notopterygium incisum seeds: the notopterygium seeds are firstly layered with wet sand at 25 ℃ for 3 months, then layered with wet sand at 4 ℃ for 3 months, taken out, washed clean with distilled water, and placed in a culture dish paved with filter paper for germination experiments in a constant-temperature illumination incubator with the temperature of 15 ℃, the humidity of 75%, the illumination intensity of 2200Lux and the illumination period of 12h (light)/12 h (dark). After 1 month, the ungerminated seeds were collected, mixed well and placed on moist sterile filter paper, which was stored in a refrigerator at 4 ℃.
Step 2, selecting and treating notopterygium seed explants: selecting healthy seeds, removing the testa of the seeds, soaking for 1min with soap water, and washing for 30min under running water;
step 3, sterilizing notopterygium seeds: soaking and disinfecting the notopterygium incisum seeds treated in the step 2 with 75% of ethanol for 20s on a sterile operating platform, then cleaning with sterile water for 2 times, then soaking and disinfecting with 0.1% of mercuric chloride for 9min, and then cleaning with sterile water for 6 times to obtain sterile seeds;
step 4, aseptically germinating notopterygium seeds: placing the sterile seeds obtained in the step 3 on sterile filter paper, transversely cutting the seeds into two halves along the direction vertical to the ventral suture line of the seeds by using a scalpel, and inoculating the seeds into a culture medium, wherein the germination culture medium comprises: MS culture medium, 1.0 mg/L6-benzylpurine (6-BA), 2.0mg/L alpha-Naphthalene Acetic Acid (NAA), 8g/L agar and 30g/L sucrose; the culture conditions of the germination culture medium are that the pH is 5.8, the temperature is 23 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 12h (light)/12 h (dark);
step 5, cluster seedling induction of aseptic seedlings: and (3) cutting off cotyledons on two sides of the sterile seedling of the seed germinated in the step (4) in a sterile operating platform, and transferring the sterile seedling into a cluster seedling induction culture medium, wherein the cluster seedling induction culture medium comprises: 1/2MS culture medium, 1.0 mg/L6-benzylpurine (6-BA), 0.6mg/L indolebutyric acid (IBA), 0.2mg/L gibberellin (GA3), 8g/L agar, 30g/L sucrose; the culture conditions of the germination culture medium are that the pH is 5.8, the temperature is 23 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 12h (light)/12 h (dark);
step 6, rooting induction of the tissue culture seedlings: dividing the cluster seedlings induced by the aseptic seedlings in the step 5 into single plants (each plant keeps about 3 leaves) in an aseptic operation table, and inoculating the single plants into a root system induction culture medium, wherein the root system induction culture medium comprises: 1/2MS culture medium, 0.6mg/L indolebutyric acid (IBA), 8g/L agar, 20g/L sucrose; the culture conditions of the germination culture medium are that the pH is 5.8, the temperature is 23 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 12h (light)/12 h (dark);
example 4 sterile Germination and Rapid propagation of Notopterygium incisum seeds
Step 1, pretreatment of notopterygium incisum seeds: the notopterygium seeds are firstly layered with wet sand at 25 ℃ for 3 months, then layered with wet sand at 4 ℃ for 3 months, taken out, washed clean with distilled water, and placed in a culture dish paved with filter paper for germination experiments in a constant-temperature illumination incubator with the temperature of 15 ℃, the humidity of 75%, the illumination intensity of 2200Lux and the illumination period of 12h (light)/12 h (dark). After 1 month, the ungerminated seeds were collected, mixed well and placed on moist sterile filter paper, which was stored in a refrigerator at 4 ℃.
Step 2, selecting and treating notopterygium seed explants: selecting healthy seeds, removing the testa of the seeds, soaking for 1min with soap water, and washing for 30min under running water;
step 3, sterilizing notopterygium seeds: soaking and disinfecting the notopterygium incisum seeds treated in the step 2 with 75% of ethanol for 20s on a sterile operating platform, then cleaning with sterile water for 2 times, then soaking and disinfecting with 0.1% of mercuric chloride for 9min, and then cleaning with sterile water for 6 times to obtain sterile seeds;
step 4, aseptically germinating notopterygium seeds: placing the sterile seeds obtained in the step 3 on sterile filter paper, cutting a wound on one side edge by using a scalpel, and inoculating the damaged seeds into a culture medium, wherein the germination culture medium comprises: MS culture medium, 0.1 mg/L2, 4-Epibrassinolide (EBR), 8g/L agar and 30g/L sucrose; the culture conditions of the germination culture medium are that the pH is 5.8, the temperature is 16 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 12h (light)/12 h (dark);
step 5, culturing the clumped seedlings of the aseptic seedlings: and (3) cutting off cotyledons on two sides of the sterile seedling of the seed germinated in the step (4) in a sterile operating platform, and transferring the sterile seedling into a cluster seedling induction culture medium, wherein the cluster seedling induction culture medium comprises: 1/2MS culture medium, 1.5 mg/L6-benzylpurine (6-BA), 0.6mg/L indolebutyric acid (IBA), 2.0mg/L alpha-naphthylacetic acid (NAA), 0.2mg/L gibberellin (GA3), 8g/L agar, 30g/L sucrose; the culture conditions of the germination culture medium are that the pH is 5.8, the temperature is 23 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 12h (light)/12 h (dark);
step 6, rooting induction of the tissue culture seedlings: dividing the cluster seedlings induced by the aseptic seedlings in the step 5 into single plants (each plant keeps about 3 leaves) in an aseptic operation table, and inoculating the single plants into a root system induction culture medium, wherein the root system induction culture medium comprises: 1/2MS culture medium, 0.6mg/L indolebutyric acid (IBA), 0.2mg/L alpha-naphthylacetic acid (NAA), 8g/L agar, 20g/L sucrose; the culture conditions of the germination culture medium are that the pH is 5.8, the temperature is 23 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 12h (light)/12 h (dark);
example 5 sterile Germination and Rapid propagation of Notopterygium incisum seeds
Step 1, pretreatment of notopterygium incisum seeds: the notopterygium seeds are firstly layered with wet sand at 25 ℃ for 3 months, then layered with wet sand at 4 ℃ for 3 months, taken out, washed clean with distilled water, and placed in a culture dish paved with filter paper for germination experiments in a constant-temperature illumination incubator with the temperature of 15 ℃, the humidity of 75%, the illumination intensity of 2200Lux and the illumination period of 12h (light)/12 h (dark). After 1 month, the ungerminated seeds were collected, mixed well and placed on moist sterile filter paper, which was stored in a refrigerator at 4 ℃
Step 2, selecting and treating notopterygium seed explants: selecting healthy seeds, removing the testa of the seeds, soaking for 1min with soap water, and washing for 30min under running water;
step 3, sterilizing notopterygium seeds: soaking and disinfecting the notopterygium incisum seeds treated in the step 2 with 75% of ethanol for 20s on a sterile operating platform, then cleaning with sterile water for 2 times, then soaking and disinfecting with 0.1% of mercuric chloride for 9min, and then cleaning with sterile water for 5 times to obtain sterile seeds;
step 4, aseptically germinating notopterygium seeds: placing the sterile seeds obtained in the step 3 on sterile filter paper, cutting a wound on one side edge by using a scalpel, and inoculating the damaged seeds into a culture medium, wherein the germination culture medium comprises: MS culture medium, 1.0 mg/L6-benzylpurine (6-BA), 3.0mg/L alpha-naphthylacetic acid (NAA), 0.2mg/L gibberellin (GA3), 8g/L agar, 30g/L sucrose; the culture conditions of the germination culture medium are that the pH is 5.8, the temperature is 23 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 12h (light)/12 h (dark);
step 5, cluster seedling induction of aseptic seedlings: and (3) cutting off cotyledons on two sides of the sterile seedling of the seed germinated in the step (4) in a sterile operating platform, and transferring the sterile seedling into a cluster seedling induction culture medium, wherein the cluster seedling induction culture medium comprises: 1/2MS culture medium, 1.5 mg/L6-benzylpurine (6-BA), 0.6mg/L indolebutyric acid (IBA), 0.2mg/L gibberellin (GA3), 8g/L agar, 30g/L sucrose; the culture conditions of the germination culture medium are that the pH is 5.8, the temperature is 23 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 12h (light)/12 h (dark);
step 6, rooting induction of the tissue culture seedlings: dividing the cluster seedlings induced by the aseptic seedlings in the step 5 into single plants (each plant keeps about 3 leaves) in an aseptic operation table, and inoculating the single plants into a root system induction culture medium, wherein the root system induction culture medium comprises: 1/2MS culture medium, 0.6mg/L indolebutyric acid (IBA), 0.2mg/L alpha-naphthylacetic acid (NAA), 8g/L agar, 20g/L sucrose; the culture conditions of the germination culture medium are that the pH is 5.8, the temperature is 23 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 12h (light)/12 h (dark).
Example 6 aseptic Germination and Rapid propagation of Notopterygium incisum seeds of the present invention
Step 1, pretreatment of notopterygium incisum seeds: the notopterygium seeds are firstly layered with wet sand at 25 ℃ for 3 months, then layered with wet sand at 4 ℃ for 3 months, taken out, washed clean with distilled water, and placed in a culture dish paved with filter paper for germination experiments in a constant-temperature illumination incubator with the temperature of 15 ℃, the humidity of 75%, the illumination intensity of 2200Lux and the illumination period of 12h (light)/12 h (dark). After 1 month, collecting ungerminated seeds, mixing well, placing on moist sterile filter paper, and storing in a refrigerator at 4 deg.C;
step 2, selecting and treating notopterygium seed explants: selecting healthy seeds, removing the testa of the seeds, soaking for 1min with soap water, and washing for 30min under running water;
step 3, sterilizing notopterygium seeds: soaking and disinfecting the notopterygium incisum seeds treated in the step 2 with 75% of ethanol for 20s on a sterile operating platform, then cleaning with sterile water for 2 times, then soaking and disinfecting with 0.1% of mercuric chloride for 9min, and then cleaning with sterile water for 5 times to obtain sterile seeds;
step 4, aseptically germinating notopterygium seeds: placing the sterile seeds obtained in the step 3 on sterile filter paper, cutting a wound on one side edge by using a scalpel, and inoculating the damaged seeds into a culture medium, wherein the germination culture medium comprises: MS culture medium, 0.1 mg/L2, 4-Epibrassinolide (EBR), 8g/L agar and 30g/L sucrose; the culture conditions of the germination culture medium are that the pH is 5.8, the temperature is 23 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 12h (light)/12 h (dark);
step 5, cluster seedling induction of aseptic seedlings: and (3) cutting off cotyledons on two sides of the sterile seedling of the seed germinated in the step (4) in a sterile operating platform, and transferring the sterile seedling into a cluster seedling induction culture medium, wherein the cluster seedling induction culture medium comprises: 1/2MS culture medium, 1.5 mg/L6-benzylpurine (6-BA), 0.6mg/L indolebutyric acid (IBA), 0.2mg/L gibberellin (GA3), 8g/L agar, 30g/L sucrose; the culture conditions of the germination culture medium are that the pH is 5.8, the temperature is 23 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 12h (light)/12 h (dark);
step 6, rooting induction of the tissue culture seedlings: dividing the cluster seedlings induced by the aseptic seedlings in the step 5 into single plants (each plant keeps about 3 leaves) in an aseptic operation table, and inoculating the single plants into a root system induction culture medium, wherein the root system induction culture medium comprises: 1/2MS culture medium, 0.6mg/L indolebutyric acid (IBA), 0.2mg/L alpha-naphthylacetic acid (NAA), 8g/L agar, 20g/L sucrose; the culture conditions of the germination culture medium are that the pH is 5.8, the temperature is 23 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 12h (light)/12 h (dark).
The following test examples illustrate the advantageous effects of the present invention:
experimental example 1 tissue culture, propagation and culture results of the present invention
1. Tissue culture results of example 1
Recovering ungerminated Notopterygii rhizoma seeds on the filter paper, cutting one side edge with a scalpel to form a wound, germinating from the cut on 3 days after inoculating into the culture medium, with germination rate of 30.48% in 1 week and maximum germination rate of 40.95% in 2 weeks. The multiplication coefficient of the Notopterygium forbesii cluster seedlings obtained in the cluster bud induction culture medium by single culture can reach 4.76 on average.
After the plants are divided, the notopterygium root seedlings are subjected to strong seedling rooting culture in a rooting culture medium, the root induction rate reaches 15.16%, the roots are strong and divergent, the tissue culture seedlings which do not root are good in growth condition, and part of the tissue culture seedlings continue to be divided into cluster seedlings.
2. Tissue culture results of example 2
Recovering ungerminated Notopterygii rhizoma seed on the filter paper, cutting the seed into two halves along the ventral suture line of the seed with a scalpel, germinating from the incision starting on day 3 of inoculating the culture medium, with germination rate of 15.24% at week 1 and maximum germination rate of 19.05% at week 2. The multiplication coefficient of the Notopterygium forbesii cluster seedlings obtained in the cluster bud induction culture medium by single culture can reach 3.94 on average.
After the plants are divided, the notopterygium root seedlings are subjected to strong seedling rooting culture in a rooting culture medium, the root induction rate reaches 15.16%, the roots are strong and divergent, the tissue culture seedlings which do not root are good in growth condition, and part of the tissue culture seedlings continue to be divided into cluster seedlings.
3. Tissue culture results of example 3
Recovering ungerminated Notopterygii rhizoma seeds on the filter paper, transversely cutting the seeds into two halves by a scalpel in the direction perpendicular to the ventral suture line of the seeds, germinating from the incision on the 3 rd day after inoculating the culture medium, wherein the germination rate of 1 week is up to 13.81%, and the germination rate of 2 week is up to the maximum of 16.19%. The multiplication coefficient of the Notopterygium forbesii cluster seedlings obtained in the cluster bud induction culture medium by single culture can reach 2.42 on average.
After the plants are divided, the notopterygium root seedlings are subjected to strong seedling rooting culture in a rooting culture medium, the root induction rate reaches 15.16%, the roots are strong and divergent, the tissue culture seedlings which do not root are good in growth condition, and part of the tissue culture seedlings continue to be divided into cluster seedlings.
4. Tissue culture results of example 4
Recovering ungerminated Notopterygii rhizoma seeds on the filter paper, cutting one side edge with a scalpel to obtain a cut, germinating from the cut on 3 days after inoculating into the culture medium, and germinating rate of 1 week is 70.48%. The multiplication coefficient of the Notopterygium forbesii cluster seedlings obtained in the cluster bud induction culture medium by single culture can reach 5.06 on average.
After the plants are divided, the notopterygium root seedlings are subjected to strong seedling rooting culture in a rooting culture medium, the root induction rate reaches 17.39%, the roots are strong and divergent, the tissue culture seedlings which do not root are good in growth condition, and part of the tissue culture seedlings continue to be divided into cluster seedlings.
5. Tissue culture results of example 5
Recovering ungerminated Notopterygii rhizoma seeds on the filter paper, cutting one side edge with a scalpel to obtain a cut, germinating from the cut on 3 days after inoculating into the culture medium, and germinating at 1 week to 65.72%. The Notopterygium forbesii cluster seedlings are obtained in the cluster seedling induction culture medium, and the multiplication coefficient of single culture can reach 5.62 on average.
After the plants are divided, the notopterygium root seedlings are subjected to strong seedling rooting culture in a rooting culture medium, the root induction rate reaches 17.39%, the roots are strong and divergent, the tissue culture seedlings which do not root are good in growth condition, and part of the tissue culture seedlings continue to be divided into cluster seedlings.
6. Example 6 tissue culture and cultivation results after transplantation of tissue culture seedlings
Selecting healthy notopterygium incisum seeds, sterilizing, and then adopting a side cutting mode to culture the optimum germination culture medium in MS +0.1mg/L EBR +30g/L sucrose +8g/L agar for 7d, wherein the germination rate is 70.48%. In the traditional germination mode, the radicle of notopterygium root breaks through the seed coat firstly, and then the cotyledon breaks through the seed coat; in this culture, however, cotyledons first grow out from the wounded area (see FIG. 1a, wherein FIGS. 1b and 1c are cross-cut and longitudinal-cut for comparison with the side-cut), and then the radicle is detached from the endosperm. After culturing the aseptic seedling for 10 days, transferring the seedling into a cluster bud induction culture medium to induce cluster buds, and culturing the seedling in the culture medium 1/2MS +1.5mg/L6-BA +0.6mg/LIBA +0.2mg/LGA3+30g/L sucrose +8g/L agar for 30 days. During the process of culturing cluster buds, it can be found that cotyledons gradually wither and die, the base parts of aseptic seedlings expand to form callus, the cluster buds gradually grow from the base parts, when the cluster buds grow initially, tissue culture seedlings are tender and delicate, leaves are curled, thin and cannot see the leaf shapes (figure 2a), as the culture time is prolonged, stems extend, the leaves begin to extend, leaf veins can be obviously observed, the edges of the leaves are notched, the leaves are slightly cracked to be feather-shaped and deeply cracked (figure 2b), and the average multiplication coefficient of single culture is 5.62. Selecting a tissue culture seedling with better growth vigor, and transferring the tissue culture seedling into an optimal rooting culture medium 1/2MS +0.6mg/LIBA +0.2mg/L NAA +20g/L sucrose +8g/L agar for rooting induction, wherein the culture period is 30 days. When the tissue culture seedlings are transferred into a rooting culture medium, about 12 days later, the tissue culture seedlings start to root, in the initial rooting process, the number of root systems is large, but the root systems grow slowly and are thick and short, the root systems grow along with the extension of the culture time, the root systems are thicker and thicker on the ground part and thinner on the ground part (figure 2c), and the root induction rate reaches 17.39%. The seedling rate of the tissue culture seedling is 95 percent along with the growth of overground parts and the growth of new buds in the rooting process. The shortest culture period is 70d, and the refining can be carried outAnd (5) transplanting seedlings. Selecting tissue culture seedlings with good growth vigor and good root growth conditions for hardening and transplanting, wherein soil for hardening and transplanting the seedlings is peat soil imported from the Netherlands, mixing the peat soil with 1/2MS +0.4mg/L NAA +0.4g/L carbendazim before transplanting, after the tissue culture seedlings are transplanted into a seedling tray filled with the peat soil, thoroughly watering the soil with 1/2MS +0.4mg/L NAA +0.4g/L carbendazim, and then culturing in an illumination incubator, wherein the temperature of the incubator is 18 +/-2 ℃, the illumination period is 12h (light)/12 h (dark), and the illumination intensity is 2500-3000 Lux. In the seedling hardening and transplanting process, the soil moisture cannot be too much, the contamination condition should be observed, and when the contamination of the soil or the tissue culture seedlings occurs, carbendazim with the concentration of 0.4g/L can be sprayed for sterilization. After the tissue culture seedlings are transplanted into the soil, as the culture time is prolonged, the stems are elongated and thickened, the leaves are stretched and thickened, and when the soil is ripped open, new sprouts grow out of the soil (fig. 2 d). The hardening seedling transplanting period is 30d, and the survival rate of the hardening seedling transplanting is 95.83 percent.
In conclusion, the tissue culture breeding method of the notopterygium incisum seeds uses the notopterygium incisum seeds which do not germinate after dormancy breaking, so that the utilization rate of the notopterygium incisum seeds is improved, and resource waste is avoided; mechanical damage is adopted to treat the seeds, so that the germination time of the seeds is shortened, and the germination rate of the seeds is improved; the germination rate is improved to more than 70 percent by culturing in a germination culture medium; the cluster seedlings can be effectively induced and differentiated in the cluster seedling induction culture medium, and the propagation coefficient of the aseptic seedlings is improved to more than 5; the root induction rate is improved to more than 17 percent by culturing in a rooting culture medium, the root system is strong and divergent, the tissue culture seedling which does not root well grows, and part of the tissue culture seedling continues to differentiate into clump seedlings. In the culture process, overground parts of notopterygium roots grow well, the seedling rate is as high as 95%, and later-period tests prove that the survival rate of the tissue culture seedlings obtained by the notopterygium root seed tissue culture breeding method after transplantation reaches over 90%, so that a solid foundation is laid for large-scale propagation of notopterygium roots, and the method has popularization and application values.
Claims (10)
1. A culture medium for tissue culture and breeding of notopterygium incisum seeds is characterized in that: the method comprises a germination culture medium, a cluster seedling induction culture medium and a root system induction culture medium;
the germination culture medium is a culture medium which takes an MS culture medium as a basic culture medium, is added with a plant growth regulator with the final concentration of 0.1-4.2 mg/L, agar with the final concentration of 5-10 g/L and cane sugar with the final concentration of 25-35 g/L, and has the pH value of 5.5-6.0;
the cluster seedling induction culture medium is a culture medium which takes 1/2MS as a basic culture medium, is added with a plant growth regulator with the final concentration of 1-4.5 mg/L, agar with the final concentration of 5-10 g/L and sucrose with the final concentration of 25-35 g/L, and has the pH value of 5.5-6.0;
the root system induction culture medium is a culture medium which takes 1/2MS culture medium as a basic culture medium, is added with plant growth regulator with final concentration of 0.4-1 mg/L, agar with final concentration of 5-10 g/L and sucrose with final concentration of 15-25 g/L, and has pH value of 5.5-6.0.
2. The culture medium according to claim 1, wherein: the plant growth regulator is any one or more of 6-benzyl purine, alpha-naphthylacetic acid, indolebutyric acid, gibberellin and 2, 4-epibrassinolide.
3. The culture medium according to claim 2, wherein: the germination culture medium is a culture medium which takes an MS culture medium as a basic culture medium, is added with 6-benzylpurine with the final concentration of 1.0mg/L, alpha-naphthylacetic acid with the final concentration of 2.0mg/L, agar with the final concentration of 8g/L and cane sugar with the final concentration of 30g/L and has the pH value of 5.8;
the clump seedling induction culture medium is a culture medium with 1/2MS as a basic culture medium, and is added with 6-benzylpurine with final concentration of 0.3-1 mg/L, indolebutyric acid with final concentration of 0.6mg/L, gibberellin with final concentration of 0.2mg/L, agar with final concentration of 8g/L and sucrose with final concentration of 30g/L, and the pH value is 5.8;
the root system induction culture medium is a culture medium which takes 1/2MS culture medium as a basic culture medium, is added with indolebutyric acid with the final concentration of 0.6mg/L, agar with the final concentration of 8g/L and cane sugar with the final concentration of 20g/L, and has the pH value of 5.5-6.0.
4. The culture medium according to claim 2, wherein: the germination culture medium is a culture medium which takes an MS culture medium as a basic culture medium, is added with 2, 4-epibrassinolide with the final concentration of 0.1mg/L, agar with the final concentration of 8g/L and sucrose with the final concentration of 30g/L and has the pH value of 5.8;
the clump seedling induction culture medium is a culture medium with 1/2MS as a basic culture medium, added with 6-benzylpurine with the final concentration of 1.5mg/L, alpha-naphthylacetic acid with the final concentration of 2.0mg/L, indolebutyric acid with the final concentration of 0.6mg/L, gibberellin with the final concentration of 0.2mg/L, agar with the final concentration of 8g/L and cane sugar with the final concentration of 30g/L, and the pH value is 5.8;
the root system induction culture medium is a culture medium which takes 1/2MS culture medium as a basic culture medium, is added with indolebutyric acid with the final concentration of 0.6mg/L, alpha-naphthylacetic acid with the final concentration of 0.2mg/L, agar with the final concentration of 8g/L and cane sugar with the final concentration of 20g/L, and has the pH value of 5.5-6.0.
5. The culture medium according to claim 2, wherein: the germination culture medium is a culture medium with the pH value of 5.8, wherein the MS culture medium is used as a basic culture medium, and is added with 6-benzylpurine with the final concentration of 1.0mg/L, 3.0mg/L alpha-naphthylacetic acid, 0.2mg/L gibberellin, 8g/L agar and 30g/L sucrose;
the clump shoot induction culture medium is a culture medium with 1/2MS as a basic culture medium, added with 6-benzylpurine with the final concentration of 1.5mg/L, indolebutyric acid with the final concentration of 0.6mg/L, gibberellin with the final concentration of 0.2mg/L, agar with the final concentration of 8g/L and cane sugar with the final concentration of 30g/L, and the pH value is 5.8;
the root system induction culture medium is a culture medium which takes 1/2MS culture medium as a basic culture medium, is added with indolebutyric acid with the final concentration of 0.6mg/L, alpha-naphthylacetic acid with the final concentration of 0.2mg/L, agar with the final concentration of 8g/L and cane sugar with the final concentration of 20g/L, and has the pH value of 5.5-6.0.
6. The culture medium according to claim 2, wherein: the germination culture medium is a culture medium which takes an MS culture medium as a basic culture medium, is added with 2, 4-epibrassinolide with the final concentration of 0.1mg/L, agar with the final concentration of 8g/L and sucrose with the final concentration of 30g/L and has the pH value of 5.8;
the clump shoot induction culture medium is a culture medium with 1/2MS as a basic culture medium, added with 6-benzylpurine with the final concentration of 1.5mg/L, indolebutyric acid with the final concentration of 0.6mg/L, gibberellin with the final concentration of 0.2mg/L, agar with the final concentration of 8g/L and cane sugar with the final concentration of 30g/L, and the pH value is 5.8;
the root system induction culture medium is a culture medium which takes 1/2MS culture medium as a basic culture medium, is added with indolebutyric acid with the final concentration of 0.6mg/L, alpha-naphthylacetic acid with the final concentration of 0.2mg/L, agar with the final concentration of 8g/L and cane sugar with the final concentration of 20g/L, and has the pH value of 5.5-6.0.
7. A tissue culture breeding method of notopterygium incisum seeds is characterized by comprising the following steps: the method is characterized by utilizing the culture medium of any one of claims 1-6 for cultivation, and comprises the following specific steps:
a. taking notopterygium incisum seeds, stacking in wet sand at 20-30 ℃ for 2-4 months, stacking in wet sand at 2-8 ℃ for 2-4 months, cleaning, germinating for 1-2 months, taking non-germinated plump seeds, stripping episperm, cleaning, and sterilizing to obtain sterile seeds
b. Taking sterile seeds, cutting out wounds, inoculating the sterile seeds into a germination culture medium, and culturing to obtain sterile seedlings;
c. taking the aseptic seedlings in the step b, cutting off cotyledons, and then transferring the aseptic seedlings into a clump seedling induction culture medium for culture to obtain clump seedlings;
d. dividing the cluster seedlings into single plants, inoculating the single plants into a root system induction culture medium, and culturing to obtain notopterygium root tissue culture seedlings.
8. The tissue culture propagation method of notopterygium incisum seeds according to claim 7, which is characterized in that: step a, cleaning and then disinfecting, namely soaking the fabric in soap water for 1min, washing the fabric with running water for 30min, finally soaking the fabric in 75% ethanol for 20s, cleaning the fabric with sterile water, soaking the fabric in 0.1% mercuric chloride for 9min, and cleaning the fabric with sterile water; the germination is carried out for 1 month under the conditions that the temperature is 15 ℃, the humidity is 75%, the illumination intensity is 2200Lux, and the illumination period is 12h (light)/12 h (dark); the layers were stratified for 3 months.
9. The tissue culture propagation method of notopterygium incisum seeds according to claim 7, which is characterized in that: step b, the wound is arranged at one side of the edge of the seed; and d, reserving 2-4 leaves on the single plant.
10. The tissue culture propagation method of notopterygium incisum seeds according to claim 7, which is characterized in that: the culture conditions are as follows: the temperature is 23 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 12h (light)/12 h (dark).
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| CN102972301A (en) * | 2012-12-08 | 2013-03-20 | 四川农业大学 | Tissue-culture seedling raising method for red birch |
| CN108719072A (en) * | 2018-06-21 | 2018-11-02 | 四川千草生物技术股份有限公司 | Rhizoma Et Radix Notopterygii seed tissue culture fast breeding technology |
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